This invention was made with government support under grant R29DK48748 awarded by the National Institutes of Health. The government has certain rights in the invention.
1. Field of the Invention
This invention relates to a member of the guanylate-binding protein family designated as GBP-4 and the cloning and expression of nucleic acid encoding this protein. The invention further relates to methods of production of the isolated molecule and its uses.
2. Description of Related Disclosures
One approach to understanding the molecular basis of cancer is to identify differences in gene expression between cancer cells and normal cells. Strategies based on assumptions that steady-state mRNA levels will differ between normal and malignant cells have been used to clone differentially expressed genes. Zhang et al., Science, 276: 1268-1272 (1997). The recent development and successful application of such strategies include the use of representational difference analysis (Braun et al., Mol Cell Biol 15: 4623-4630 (1995); Lewis et al., Mol Cell Biol 17: 4967-4978 (1997)), serial analysis of gene expression (Velculescu et al., Science, 270: 484-487 (1995)), and quantitative hybridization analysis of arrayed cDNA""s. Schena et al., Science, 270: 467-470 (1995).
Cancer of the stomach is a leading cause of cancer deaths worldwide. Parsonnet, Gastroenterol Clin North Am, 22: 89-104 (1993); Peddanna et al., Anticancer Res, 15: 2055-64 (1995). The development of intestinal-type gastric cancer is characterized by successive histopathologic changes progressing from normal mucosa to gastritis to metaplasia and eventually to dysplasia. Stemmermann et al., Hum Pathol, 25: 968-981 (1994).
Cells treated with interferons respond to this stimulus by producing a set of proteins believed to serve as intracellular mediators of the various effects of these cytokines. Interferon-induced GBP""s are members of the G protein superfamily and form a distinct subgroup based on their large size (65-67 kDa), potent induction of interferon-xcex3, and relaxed nucleotide binding. specificity. Nantais et al., J Leukoc Biol, 60: 423-431 (1996). Two other subgroups of GTP binding proteins which are induced by interferon include the Mx proteins, which are involved in the antiviral response (Horisberger et al., J Virol, 64:1171-81 (1990)), and the inducibly expressed GTPases (IGTP""s) which are endoplasmic reticulum GTPases that may be involved in protein processing or trafficking. Taylor et al., J. Biol Chem, 271: 20399-20405 (1996); Taylor et al., J Biol Chem, 272: 10639-10645 (1997). Interferon-induced GBP""s are GTPases which hydrolyze GTP to GDP and at least one GBP, and can bind to agarose-immobilized guanine nucleotides. Cheng et al., J.Biol. Chem., 258: 7746-7750 (1983). GBPs bind to GMP and GTP with similar affinity. Cheng et al., Mol Cell Biol, 11: 4717-4725 (1991); Schwemmle et al., J Biol Chem, 271: 10304-10308 (1996); Schwemmle and Staeheli, Curr Opin Cell Biol, 6: 253-259 (1994). cDNAs for chicken, rat, mouse and human GBPs have been isolated. Asundi et al., Biochim Biophys Acta, 1217:257-265 (1994); Cheng et al., (1991), supra; Schwemmle et al., J Biol Chem, 271: 10304-10308 (1996); Wynn et al., J Immunol, 147: 4384-4392 (1991). Purification of a GBP is described in Cheng et al., J. Biol. Chem., 260: 15834-15839 (1985). It was later found that two distinct interferon-alpha and -gamma-inducible genes code for two human GBPs, designated hGBP1 and hGBP2. Cheng et al., (1991), supra. Subsequently, human GBP3 was identified and partially characterized. Strehlow et al., Gene, 144: 295-299 (1994). GBP-3 was shown to have a structure related to GBP-1 and a high degree of sequence homology to both GBP-1 and GBP-2.
Schwemmle and Staeheli, J. Biol. Chem., 269: 11299-11305 (1994) showed that hGBP1, the human 67-kDa guanylate-binding protein, is a GTPase that converts GTP to GMP. Since GTP analogs with a cleavage-resistant bond between the beta- and gamma-phosphates could not be hydrolyzed by hGBP1, and pyrophosphate was no reaction product, hGBP1 seemed to degrade GTP by two consecutive cleavages of single phosphate groups. They further showed that it can be isoprenylated in vitro.
Interferon-induced GBPs have been isolated from other species such as rat (Asundi et al., Biochim. Biophys. Acta, 1217: 257-265 (1994); Vestal et al., Biochem. Biophys. Res. Commun., 224: 528-534 (1996)), murine (Vestal et al., Molecular Bioloay of the Cell, 7 (SUPPL.): 527A (1996) presented at the Annual Meeting of the 6th International Congress on Cell Biology and the 36th American Society for Cell Biology, San Francisco, Calif., USA, Dec. 7-11, 1996; Vestal et al., Molecular Biology of the Cell, 6 (SUPPL.): 288A (1995) presented at the Thirty-fifth Annual Meeting of the American Society for Cell Biology, Washington, D.C., USA, Dec. 9-13, 1995), and chicken. Schwemmle et al., J. Biol. Chem., 271: 10304-10308 (1996). Sequence analysis revealed that human and mouse GBPs contain only the first two elements of the typical GTP-binding consensus motif and that they contain the same sequence motif at their C termini as p21ras. Cheng et al., (1991), supra. The predicted protein sequences of these interferon-induced GBPs lack one of the three sequence motifs typically found in GTP/GDP binding proteins. The GBPs also contain carboxyl terminal residues which are substrates for post-translational modification by protein-prenyl transferases. Nantais et al., J Leukoc Biol, 60: 423-431 (1996). Members of the Ras superfamily of GTPases are prenylated and the prenyl modification of these proteins is required for their biological function and cellular localization. Zhang and Casey, Annu Rev Biochem, 65: 241-269 (1996).
GTPases serve many different cellular functions. For example, they play key roles in such basic processes as signal transduction, vesicle transport, and translation. Most GTPases contain a tripartite GTP-binding consensus motif. Dever et al., Proc. Natl. Acad. Sci. USA, 84: 1814-1818 (1987). These sequences form part of the GTP binding pocket. The typical GTPases can assume two distinct conformations, a GTP-bound (active) and a GDP-bound (inactive) conformation. Hence, they can potentially function as molecular switches. Some GTPases, like p21ras and heterotrimeric G proteins, have a sequence motif at their C-termini that functions as an isoprenylation signal and thus ensures the proper anchoring of these proteins in cell membranes.
The histopathologic observation by Stemmermann et al., supra, that gastric cancer progresses from normal through to dysplasia prompted investigation into the molecular events which govern this progression from gastritis to malignancy. The study of tumor pathogenesis herein was begun by isolating RNA from normal stomach and from a gastric adenocarcinoma and applying a suppression subtractive hybridization (SSH) technique discussed below. One of the differentially expressed clones identified, and then isolated and characterized, is a new member of the interferon-induced guanylate-binding protein (GBP) gene family, GBP-4.
Accordingly, this invention provides isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 95% sequence identity to (a) a DNA molecule encoding a human GBP-4 polypeptide comprising the sequence of amino acids 1-5591 of FIG. 1 (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a). Preferably, the nucleic acid has at least one GBP-4 biological activity. Also preferred is that the nucleic acid comprise DNA having at least about a 99% sequence identity to (a) a DNA molecule encoding a human GBP-4 polypeptide comprising the sequence of amino acids 1 to 591 of FIG. 1 (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a). More preferably, the nucleic acid comprises DNA encoding a human GBP-4 polypeptide having amino acid residues 1 to 591 of FIG. 1 (SEQ ID NO:3), or the complement of the encoding DNA.
In another aspect, the invention provides isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 95% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human GBP-4 polypeptide cDNA in ATCC Deposit No. 209,456 (pRK5-based plasmid pRK5.hu.GBP4-histag.71), or (b) the complement of the DNA molecule of (a).
In further embodiments of the invention, a vector is provided comprising the nucleic acid, as well as a host cell comprising the vector, especially a host cell that is a human cell, CHO cell, or E. coli. In a further aspect, the invention provides a process for producing a GBP-4 polypeptide comprising culturing the host cell under conditions suitable for expression of the GBP-4 polypeptide and recovering the GBP-4 polypeptide from the cell culture.
Also provided is isolated GBP-4 polypeptide encoded by the nucleic acid, preferably human GBP-4.
Further provided is a chimeric molecule comprising a GBP-4 polypeptide fused to a heterologous amino acid sequence. In preferred embodiments, the heterologous amino acid sequence is an epitope tag sequence or an Fc region of an immunoglobulin.
In yet a further embodiment, the invention provides an antibody which specifically binds to a GBP-4 polypeptide, preferably a monoclonal antibody.
In a still further aspect, the invention supplies isolated nucleic acid having at least about 600 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human GBP-4 polypeptide comprising the sequence of amino acids 1 to 591 of FIG. 1 (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has at least about a 95% sequence identity to (a) or (b), isolating the test DNA molecule.
Additionally, the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human GBP-4 polypeptide comprising the sequence of amino acids 1 to 591 of FIG. 1 (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about a 95% sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
In other embodiments, the invention provides a composition comprising the polypeptide and a carrier therefor, as well as a composition comprising an antagonist to the polypeptide and a carrier therefor. Preferably, the composition further comprises GTP.
In still further embodiments, the invention provides a method of determining the presence in a test sample of a molecule that binds to a guanylate binding protein comprising contacting the test sample with the polypeptide and determining if binding has occurred. In a preferred embodiment, the molecule that binds to the protein is a guanine nucleotide.
In a still further embodiment, the invention supplies a method of determining the presence in a test sample of a guanylate-binding protein-4 comprising contacting the test sample with an immobilized guanine nucleotide and determining if binding has occurred.
Additionally, the invention provides a method for purifying molecules that bind to a guanylate-binding protein comprising contacting a sample containing the molecules to be purified with the polypeptide immobilized on a support under conditions whereby the molecules to be purified are selectively adsorbed onto the immobilized protein, washing the immobilized support to remove non-adsorbed material, and separating the molecules to be purified from the immobilized protein to which they are adsorbed. Preferably, the molecules to be purified are guanine nucleotides.
Also provided is a method of amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase chain reaction with the nucleic acid disclosed above.
Further provided is a method of determining the presence of nucleic acid encoding guanylate-binding protein-4 in a test sample comprising contacting the nucleic acid disclosed above with the test sample and determining whether hybridization has occurred.
The cDNA clone encoding GBP-4 was isolated from a gastric adenocarcinoma by SSH, a technique described below. The predicted amino acid sequence of GBP-4 is homologous with that of a family of mammalian and chicken interferon-inducible GTP binding proteins. GBP-4 shares 60% overall sequence homology with the proteins encoded by cDNA""s for human GBP-1 and GBP-2. Cheng et al., (1991), supra. The molecule shares many characteristics with other family members such as chromosomal localization and interferon inducibility. The GBP-4 gene was localized to human chromosome 1p31-1p32, closely linked to the interferon-induced GBP genes, GBP-1 and GBP-2.
Immunoelectron microscopy indicated that GBP-4 was associated with the membranes of endolysosomes. GBP-4 was expressed in many normal tissues examined, with highest levels in peripheral blood leukocytes, lymph node, and the spleen. Like GBP-1 and GBP-2, GBP-4 expression was induced in human cell lines by IFN-xcex3. Unlike GBP-1 and GBP-2, however, which were both expressed in normal stomach and in stomach tumor tissue, GBP-4 expression was found only in the gastric adenocarcinoma. In situ hybridization performed on gastric tumor tissue demonstrated that GBP-4 mRNA was associated with malignant epithelial cells. The differential expression of GBP-4 and its biological characteristics suggests a role for guanylate binding proteins in tumorigenesis, as well as in inflammatory and immune responses.